1. Field of the Invention
The present invention relates to genetically engineered plants. More particularly, the present invention relates to the optimization of substrate pools to facilitate the biosynthetic production of commercially useful polyhydroxyalkanoates (PHAs) in plants.
The present invention especially relates to the production of copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV), designated P(3HB-co-3HV) copolymer, and derivatives thereof.
2. Description of Related Art
Polyhydroxyalkanoates
Polyhydroxyalkanoates are polyesters that accumulate in a wide variety of bacteria. These polymers have properties ranging from stiff and brittle plastics to rubber-like materials, and are biodegradable. Due to these properties, PHAs are an attractive source of non-polluting plastics and elastomers.
Currently, there are approximately a dozen biodegradable plastics in commercial use that possess properties suitable for producing a number of specialty and commodity products (Lindsay, 1992). One such biodegradable plastic in the polyhydroxyalkanoate (PHA) family that is commercially important is Biopol.TM., a random copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). This bioplastic is used to produce biodegradable molded material (e.g., bottles), films, coatings, and in drug release applications. Biopol.TM. is produced via a fermentation process employing the bacterium Alcaligenes eutrophus (Byrom, 1987). The current market price is $6-7/lb, and the annual production is 1,000 tons. By best estimates, this price is likely to be reduced only about 2-fold via fermentation (Poirier et al., 1995). Competitive synthetic plastics such as polypropylene and polyethylene cost about 35-45.cent./lb (Layman, 1994). The annual global demand for polyethylene alone is about 37 million metric tons (Poirier et al., 1995). It is therefore likely that the cost of producing P(3HB-co-3HV) by microbial fermentation will restrict its use to low-volume specialty applications.
Nakamura et al. (1992) reported using threonine (20 g/L) as the sole carbon source for the production of P(3HB-co-3HV) copolymer in A. eutrophus. These workers initially suggested that the copolymer might form via the degradation of threonine by threonine deaminase, with conversion of the resultant .alpha.-ketobutyrate (=2-oxobutyrate) to propionyl-CoA. However, they ultimately concluded that threonine was utilized directly, without breaking carbon-carbon bonds, to form valeryl-CoA as the 3HV precursor. The nature of this chemical conversion was not described, but since the breaking of carbon-carbon bonds was not postulated to occur, the pathway could not involve threonine deaminase in conjunction with an .alpha.-ketoacid decarboxylating step to form propionate or propionyl-CoA. In the experiments of Nakamura et al., the PHA polymer content was very low (&lt;6% of dry cell weight). This result, in conjunction with the expense of feeding bacteria threonine, makes their approach impractical for the commercial production of P(3HB-co3HV) copolymer.
Yoon et al. (1995) have shown that growth of Alcaligenes sp. SH-69 on a medium supplemented with threonine, isoleucine, or valine resulted in significant increases in the 3HV fraction of the P(3HB-co-3HV) copolymer. In addition to these amino acids, glucose (3% wt/vol) was also added to the growth media. In contrast to the results obtained by Nakamura et al. (1992), growth of A. eutrophus under the conditions described by Yoon et al. (1995) did not result in the production of P(3HB-co-3HV) copolymer when the medium was supplemented with threonine as the sole carbon source. From their results, Yoon et al. (1995) implied that the synthetic pathway for the 3HV component in P(3HB-co-3HV) copolymer is likely the same as that described in WO 91/18995 and Steinbuchel and Pieper (1992). This postulated synthetic pathway involves the degradation of isoleucine to propionyl-CoA (FIG. 3).
The PHB Biosynthetic Pathway
Polyhydroxybutyrate (PHB) was first discovered in 1926 as a constituent of the bacterium Bacillus megaterium (Lemoigne, 1926). Since then, PHAs such as PHB have been found in more than 90 different genera of gram-negative and gram-positive bacteria (Steinbuchel, 1991). These microorganisms produce PHAs using R-.beta.-hydroxyacyl-CoAs as the direct metabolic substrate for a PHA synthase, and produce polymers of R-(3)-hydroxyalkanoates having chain lengths ranging from C3-C14 (Steinbuchel and Valentin, 1995).
To date, the best understood biochemical pathway for PHB production is that found in the bacterium Alcaligenes eutrophus (Dawes and Senior, 1973; Slater et al., 1988; Schubert et al., 1988; Peoples and Sinskey, 1989a and 1989b). This pathway, which is also utilized by other microorganisms, is summarized in FIG. 1. In this organism, an operon encoding three gene products, i.e., PHB synthase, .beta.-ketothiolase, and acetoacetyl-CoA reductase, encoded by the phbC, phbA, and phbB genes, respectively, are required to produce the PHA homopolymer R-polyhydroxy-butyrate (PHB).
As further shown in FIG. 1, acetyl-CoA is the starting substrate employed in the biosynthetic pathway. This metabolite is naturally available for PHB production in the cytoplasm and plastids of plants.
Poirier et al. (1992) demonstrated that a multi-enzyme pathway can be introduced into plants to produce polyhydroxybutyrate (PHB). In that work, the genes encoding the Alcaligenes eutrophus acetoacetyl-CoA reductase (phbB) and PHB synthase (phbC) genes were introduced into Arabidopsis thaliana, where the enzymes were expressed cytoplasmically. A 3-ketothiolase is already expressed in the cytoplasm of Arabidopsis. Although PHB was produced in the plants which expressed the three enzymes, the yield was low and the plants were stunted and had reduced seed production.
Nawrath et al. (1994) provided a solution to these problems. There, the genes for the three bacterial PHB enzymes (phbC, phbA, and phbB) were modified to comprise a pea chloroplast targeting peptide (="transit peptide"), which targeted the enzymes to the chloroplast. Arabidopsis plants which produced these three enzymes in the chloroplast accumulated large amounts of PHB. There was also no apparent affect of these transgenes, or of the PHB accumulation, on the growth and development of the transgenic plants.
The P(3HB-co-3HV) Copolymer Biosynthetic Pathway
As noted above, P(3HB-co-3HV) random copolymer, commercially known as Biopol.TM., is produced by fermentation employing A. eutrophus. A proposed biosynthetic pathway for P(3HB-co-3HV) copolymer production is shown in FIG. 2. Production of this polymer in plants has been reported (oral presentation by Mitsky et al., 1997).
Since the production of PHB in chloroplasts apparently does not affect plant growth and development as does production of PHB in the cytoplasm (Nawrath et al., 1992), the chloroplast is the preferred site of P(3HB-co-3HV) biosynthesis. The successful production of P(3HB-co-3HV) copolymer in plants thus requires the presence of three PHA biosynthetic enzymes as well as the substrates required for the copolymer biosynthesis (FIG. 2), preferably in the plastids. For the 3HB component of the polymer, the substrate naturally exists in chloroplasts in sufficient concentration in the form of acetyl-CoA (Nawrath et al., 1994). However, this is not true for the 3HV component of the polymer, where the starting substrate is propionyl-CoA. FIG. 3 is an overview of enzyme pathways which are related to the provision of these substrates. The engineering of plants to generate sufficient chloroplast pools of propionyl-CoA, along with the proper PHA biosynthetic enzymes (i.e., a .beta.-ketothiolase, a .beta.-ketoacyl-CoA reductase, and a PHA synthase), makes it possible to produce copolyesters of poly(3HB-co-3HV) in these organisms.
Methods for optimization of PHB and P(3HB-co-3HV) production in various crop plants are disclosed in Gruys et al. (1998). A major focus in that invention is the optimization of the substrate pools for P(3HB-co-3HV), in order to provide 2-ketobutyrate and propionyl-CoA to the site of copolymer synthesis. Gruys et al. (1998) also discusses exploring the potential use of a pyruvate dehydrogenase complex and a branched chain oxoacid dehydrogenase complex to convert 2-oxobutyrate to propionyl-CoA.
Gruys et al. (1998) also provides methods for the optimization of .beta.-ketothiolase, .beta.-ketoacyl-CoA reductase, and PHA synthase activities in plants and bacteria. It was determined therein that the A. eutrophus .beta.-ketothiolase PhbB was metabolically blocked from producing P(3HB-co-3HV) due to its inability to utilize propionyl-CoA with acetyl-CoA to produce 3-ketovaleryl-CoA (see FIG. 2). However, Gruys et al. (1998) demonstrated that another A. eutrophus .beta.-ketothiolase, designated BktB, is able to produce 3-ketovaleryl-CoA from propionyl-CoA and acetyl-CoA. Therefore, BktB is a preferred .beta.-ketothiolase for the production of P(3HB-co-3HV). Gruys et al. also demonstrated that other .beta.-ketothiolases are able to produce 3-keto-valeryl-CoA from propionyl-CoA and acetyl-CoA. These are: another A. eutrophus .beta.-ketothiolase, designated pAE65, and two .beta.-ketothiolases from Zoogloea ramigera, designated "A" and "B".
Gruys et al. (1998) noted that the sources of the three copolymer biosynthetic enzymes may encompass a wide range of organisms, including, for example, Alcaligenes eutrophus, Alcaligenes faecalis, Aphanothece sp., Azotobacter vinelandii, Bacillus cereus, Bacillus megaterium, Beijerinkia indica, Derxia gummosa, Methylobacterium sp., Microcoleus sp., Nocardia corallina, Pseudomonas cepacia, Pseudomonas extorguens, Pseudomonas oleovorans, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum (Brandl et al., 1990; Doi, 1990), and Thiocapsa pfennigii.
Pyruvate Dehydrogenase Complex
The pyruvate dehydrogenase complex (PDC) is a large multi-enzyme structure composed of three primary component enzymes, pyruvate dehydrogenase (PDH) (E1, EC 1.2.41); dihydrolipoamide acetyltransferase (E2, EC 2.3.1.12); and dihydrolipoamide dehydrogenase (E3, EC 1.8.1.4) (Reed, 1974). In the well-characterized mammalian complex, 60 subunits of E2 comprise the central core, and the E1 and E3 components decorate the outer surface of this core (Patel et al., 1990). E1 is a heterotetramer composed of two .alpha. and two .beta. subunits. The E3 component, a homodimer, associates with the complex via an E3 binding protein (Gopalakrishnan, 1989).
The PDC catalyzes the irreversible oxidative decarboxylation of pyruvate according to the equation: EQU Pyruvate+CoA+NAD.sup.+ .fwdarw.Acetyl-CoA+CO.sub.2 +NADH+H.sup.+
In mitochondria, this reaction represents the irreversible commitment of carbon to the citric acid cycle, and therefore is a logical point for regulation. Previous experiments have shown that plant mitochondrial PDC activity is, in fact, regulated by product inhibition, metabolites, and reversible phosphorylation (Randall et al., 1977; Randall et al., 1989; Randall et al., 1996; Budde et al, 1991) as is the mammalian complex (Patel et al., 1990).
In prokaryotes, PDC is localized in the cytoplasm, while in eukaryotes it is within the mitochondrial matrix. Plants, however, are unique in that a second form of the complex exists in the plastids (Reid et al., 1975; Reid et al., 1977; Thompson et al, 1977b). Based upon enzymology (Thompson et al., 1977a; Williams et al., 1979; Camp et al., 1988) and immunochemical analyses (Taylor et al., 1992; Camp et al, 1985) it is clear that plastid PDC is distinct from its mitochondrial counterpart. In plants, de novo fatty acid biosynthesis occurs exclusively in the plastids (Miernyk et al., 1983; Kang et al., 1994; Zilket et al., 1969; Drennan et al., 1969; Ohlrogge et al., 1979). The plastid form of PDC can provide the fatty acid precursor, acetyl-CoA (Miernyk et al., 1983; Kang et al., 1994; Grof et al., 1995). The plastid PDC can also catalyze the oxidative decarboxylation of 2-oxobutyrate to produce propionyl CoA (Camp et al., 1988; Camp and Randall, 1985).
The cDNAs that encode the E1.alpha. and E1.beta. subunits of plant mitochondrial PDH have been cloned (Grof et al., 1995; Leuthy et al., 1995; Leuthy et al, 1994). Recently, Reith and Munholland (1995) reported the sequence of the entire plastid genome of the red alga P. purpurea. Encoded in this genome are open reading frames homologous to PDH .alpha. and .beta. subunits.
The cDNAs that encode the E2 component of the plant mitochondrial PDC have been similarly cloned (Guan et al., 1995). The sequence of the entire plastid genome of the cyanobacterium Synechocystis sp. has also recently been reported (Kaneko et al., 1996).
Branched Chain 2-Oxoacid Dehydrogenase Complex
The branched chain 2-oxoacid dehydrogenase complex (BCOADC) is a highly ordered macromolecular structure composed of three primary component enzymes, a branched chain dehydrogenase or decarboxylase (BCDH or E1; EC 1.2.4.4); dihydrolipoamide transacylase (LTA or E2; no EC number); and dihydrolipoamide dehydrogenase (LipDH or E3; EC 1.8.1.4) (Yeaman, 1989). The mammalian complex is assembled with 24 subunits of E2 as the central cubic core with 4:3:2 symmetry; the E1 and E3 components decorate the outer surface of the E2 core (Yeaman, 1989; Wynn et al., 1996). E1 is a heterotetramer composed of two identical .alpha. and two identical .beta. subunits (Pettit et al., 1978). E3 associates loosely with the E2-E1 structure, and is a homodimer (Wynn et al., 1996; Pettit et al., 1978). The mammalian mitochondrial complex is also regulated by a specific E1-kinase and a phospho-E1 phosphatase, which modulate activity by reversible phosphorylation (inactivation) and dephosphorylation (reactivation). Additional regulation is achieved by product inhibition and modulation of gene expression (Yeaman, 1989; Wynn et al., 1996).
BCOADC catalyzes the irreversible oxidative decarboxylation of the branched-chain 2-oxoacids derived from valine, leucine and isoleucine, as well as 2-oxobutyrate and 4-methyl-2-oxobutyrate, with comparable rates and similar Km values (Yeaman 1989; Wynn et al., 1996; Paxton et al., 1986; Gerbling et al., 1988). The reactions are: EQU 2-oxo-3-methylvalerate+CoA+NAD.sup.+ .fwdarw.2-methylbutyryl-CoA+CO.sub.2 +NADH+H.sup.+ EQU 2-oxo-isovalerate+CoA+NAD.sup.+ .fwdarw.isobutyryl-CoA+CO.sub.2 +NADH+H.sup.+ EQU 2-oxo-isocaproiate+CoA+NAD.sup.+ .fwdarw.isovalyryl-CoA+CO.sub.2 +NADH+H.sup.+ EQU 2-oxobutyrate+CoA+NAD.sup.+ .fwdarw.propionyl-CoA+CO.sub.2 +NADH+H.sup.+
In mammals, BCOADC is found in the mitochondria and is involved in the catabolism of the branched-chain amino acids. The only reports describing BCOADC activity in plants have localized BCOADC to peroxisomes (Gerbling et al., 1988; Gerbling et al., 1989). The proposed function of a peroxisomal BCOADC is to catabolize the branched-chain amino acids during germination and growth, yielding an acyl-CoA product that would be further metabolized by the beta-oxidation pathway localized in the peroxisome (Gerbling et al., 1988; Gerbling et al., 1989).
To provide substrate pools to permit biosynthesis of P(3HB-co-3HV) copolymer in the plastid, there is a need for methods to engineer plants to produce plastid enzymes which convert 2-oxobutyrate to propionyl-CoA.